John H. Exton

Phosphatidylcholine breakdown and signal transduction.

PC hydrolysis by PLA2, PLC or PLD is a widespread response elicited by most growth factors, cytokines, neurotransmitters, hormones and other extracellular signals. The mechanisms can involve G-proteins, PKC, Ca2+ and tyrosine kinase activities. Although an agonist-responsive cytosolic PLA2 has been purified, cloned and sequenced, the agonist-responsive form(s) of PC-PLC has not been identified and no form of PC-PLD has been purified or cloned. Regulation of PLA2 by Ca2+ and MAPK is well established and involves membrane translocation and phosphorylation, respectively. PKC regulation of the enzyme in intact cells is probably mediated by MAPK. The question of G-protein control of PLA2 remains controversial since the nature of the G-protein is unknown and it is not established that its interaction with the enzyme is direct or not. Growth factor regulation of PLA2 involves tyrosine kinase activity, but not necessarily PKC. It may be mediated by MAPK. The physiological significance of PLA2 activation is undoubtedly related to the release of AA for eicosanoid production, but the LPC formed may have actions also. There is much evidence that PKC regulates PC-PLC and PC-PLD and this is probably a major mechanism by which agonists that promote PI hydrolysis secondarily activate PC hydrolysis. Since no agonist-responsive forms of either phospholipase have been isolated, it is not clear that PKC exerts its effects directly on the enzymes. Although it is assumed that a phosphorylation mechanism is involved, this may not be the case, and regulation may be by protein-protein interactions. G-protein control of PC-PLD is well-established, although, again, it has not been demonstrated that this is direct, and the nature of the G-protein(s) involved is unknown. In some cell types, there is evidence of the participation of a soluble protein, which may be a low Mr GTP-binding protein. What role this plays in the activation of PC-PLD is obscure. Agonist activation of PC hydrolysis in cells is usually Ca(2+)-dependent, but the step at which Ca2+ is involved is unclear, since PC-PLD and PC-PLC per se are not influenced by physiological concentrations of the ion. Most growth factors promote PC hydrolysis and this is mainly due to activation of PKC as a result of PI breakdown. However, in some cases, PC breakdown occurs in the absence of PI hydrolysis, implying another mechanism that does not involve PI-derived DAG.(ABSTRACT TRUNCATED AT 400 WORDS)

Autocrine Tumor Necrosis Factor Alpha Links Endoplasmic Reticulum Stress to the Membrane Death Receptor Pathway through IRE1α-Mediated NF-κB Activation and Down-Regulation of TRAF2 Expression

ABSTRACT NF-κB is critical for determining cellular sensitivity to apoptotic stimuli by regulating both mitochondrial and death receptor apoptotic pathways. The endoplasmic reticulum (ER) emerges as a new apoptotic signaling initiator. However, the mechanism by which ER stress activates NF-κB and its role in regulation of ER stress-induced cell death are largely unclear. Here, we report that, in response to ER stress, IKK forms a complex with IRE1α through the adapter protein TRAF2. ER stress-induced NF-κB activation is impaired in IRE1α knockdown cells and IRE1α−/− MEFs. We found, however, that inhibiting NF-κB significantly decreased ER stress-induced cell death in a caspase-8-dependent manner. Gene expression analysis revealed that ER stress-induced expression of tumor necrosis factor alpha (TNF-α) was IRE1α and NF-κB dependent. Blocking TNF receptor 1 signaling significantly inhibited ER stress-induced cell death. Further studies suggest that ER stress induces down-regulation of TRAF2 expression, which impairs TNF-α-induced activation of NF-κB and c-Jun N-terminal kinase and turns TNF-α from a weak to a powerful apoptosis inducer. Thus, ER stress induces two signals, namely TNF-α induction and TRAF2 down-regulation. They work in concert to amplify ER-initiated apoptotic signaling through the membrane death receptor.

Regulation of phospholipase D.

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.

A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes.

Abstract A method utilizing precipitation on filter paper has been used to isolate glycogen from homogenates of liver or isolated hepatocytes. The procedure requires very small amounts of tissue or cell suspension and is rapid and highly reproducible. Its efficiency and specificity make it very suitable for many studies involving radioactive tracer incorporation into glycogen.

Phospholipase C-β1 is a GTPase-activating protein for Gq/11, its physiologic regulator

Purified M1 muscarinic cholinergic receptor and Gq/11 were coreconstituted in lipid vesicles. Addition of purified phospholipase C-beta 1 (PLC-beta 1) further stimulated the receptor-promoted steady-state GTPase activity of Gq/11 up to 20-fold. Stimulation depended upon receptor-mediated GTP-GDP exchange. Addition of PLC-beta 1 caused a rapid burst of hydrolysis of Gq/11-bound GTP that was at least 50-fold faster than in its absence. Thus, PLC-beta 1 stimulates hydrolysis of Gq/11-bound GTP and acts as a GTPase-activating protein (GAP) for its physiologic regulator, Gq/11. GTPase-stimulating activity was specific both for PLC-beta 1 and Gq/11. Such GAP activity by an effector coupled to a trimeric G protein can reconcile slow GTP hydrolysis by pure G proteins in vitro with fast physiologic deactivation of G protein-mediated signaling.

New Developments in Phospholipase D

Phospholipase D (PLD) (1) is present in bacteria, fungi, plants, and animals. It is widely distributed in mammalian cells, where it is regulated by a variety of hormones, growth factors, and other extracellular signals. Its major substrate is phosphatidylcholine (PC), which is hydrolyzed to phosphatidic acid (PA) and choline, but it can also act on phosphatidylethanolamine and phosphatidylinositol in some organisms and tissues. It also catalyzes a phosphatidyl transfer reaction in which a primary alcohol acts as nucleophilic acceptor in place of H2O. The resulting production of phosphatidyl alcohol represents a specific assay for PLD.

Regulation of phospholipase D

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N‐terminus and have demonstrated that a conserved sequence at the C‐terminus is required for catalysis. The N‐ and C‐terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non‐phosphorylating mechanism. Small G proteins of the Rho and ADP‐ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C‐terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.

Mechanisms of action of calcium-mobilizing agonists: some variations on a young theme.

It is now accepted that many hormones and neurotransmitters exert their effects through G protein‐mediated activation of a phospholipase C, which breaks down phosphatidylinositol bisphosphate. This releases inositol trisphosphate, which mobilizes intracellular calcium, and diacylglycerol, which, in turn, activates protein kinase C. However, recent evidence indicates that other mechanisms are involved. In some cells, the increases in cytosolic calcium elicited within 1‐2 s by high concentrations of agonists or at later times by low, physiological concentrations of agonists occur without any detectable changes in inositol phosphates and calcium mobilization, and result from the opening of plasma membrane channels that are permeable to Ca2+. This response appears to be mediated more directly by G proteins. These findings question the postulated roles of inositol phosphates and calcium mobilization in the stimulation of calcium influx. Measurements of the mass and fatty acid composition of the inositol phospholipids and of the diacylglycerol and phosphatidic acid generated by agonists in several cell types indicate that phosphatidylinositol bisphosphate is probably a minor source of these lipids. On the other hand, measurements of phosphatidylcholine, choline, and phosphocholine indicate that this phospholipid is a major source, and that its breakdown involves both phospholipase C and D. These findings indicate that phosphatidylcholine breakdown may be more important than phosphoinositide hydrolysis in the regulation of protein kinase C and perhaps other cell functions.— Exton, J. H. Mechanisms of action of calcium mobilizing agonists: some variations on a young theme. FASEB J. 2: 2670‐2676; 1988.

Involvement of Tyrosine Phosphorylation and Protein Kinase C in the Activation of Phospholipase D by H2O2 in Swiss 3T3 Fibroblasts

We have investigated the mechanisms involved in H2O2-mediated phospholipase D (PLD) activation in Swiss 3T3 fibroblasts. In the presence of vanadate, H2O2 induced tyrosine phosphorylation of PLD as well as the platelet-derived growth (PDGF) factor receptor, protein kinase Cα (PKCα), and a 62-kDa protein in rat brain PLD1 (rPLD1) immune complexes. PDGF also induced tyrosine phosphorylation of PLD, but this was abolished by catalase, indicating that it was mediated by H2O2 generation. Interestingly, PLD was found to be constitutively associated with the PDGF receptor and PKCα. Stimulation by H2O2 showed a concentration- and time-dependent tyrosine phosphorylation of the proteins in rPLD1 immunoprecipitates and activation of PLD in the cells. Pretreatment of the cells with the protein-tyrosine kinase inhibitors genistein and herbimycin A resulted in a concentration-dependent inhibition of H2O2-induced tyrosine phosphorylation and PLD activation. Activation of PLD by H2O2 was also inhibited dose-dependently by the PKC inhibitors Ro 31-8220 and calphostin C. Down-regulation of PKC by prolonged treatment with 4β-phorbol 12-myristate 13-acetate also abolished H2O2-stimulated PLD activity. H2O2 or vanadate alone did not induce tyrosine phosphorylation of proteins in the rPLD1 immune complex or PLD activation. Reduction of intracellular H2O2levels by pretreatment of the cells with catalase dramatically abrogated tyrosine phosphorylation of proteins in the rPLD1 immune complex and PLD activation, suggesting the potential role of intracellular H2O2 in H2O2-mediated PLD signaling. Taken together, these results suggest that both protein-tyrosine kinase(s) and protein kinase C participate in H2O2-induced PLD activation in Swiss 3T3 cells.

THE ROLE OF CYCLIC AMP IN THE INTERACTION OF GLUCAGON AND INSULIN IN THE CONTROL OF LIVER METABOLISM

It is now well established that insulin exerts direct effects on mammalian liver to inhibit the production of glucose and urea and to promote the uptake of potassium ions. It has been proposed that these effects of insulin may be partly due to a decrease in liver cyclic AMP.l The proposal is based on the following observations: ( 1) insulin produces a small but significant decrease in the level of cyclic AMP in the perfused rat liver; (2) depletion of insulin in vivo by treatment with insulin antiserum or alloxan results in a twofold increase in liver cyclic AMP; (3) exogenous cyclic AMP and hormones such as glucagon and epinephrine which raise the level of cyclic AMP produce effects on the liver which are opposite to those caused by insulin; (4) insulin antagonizes the actions of epinephrine, glucagon, or cyclic AMP in the perfused liver; and ( 5 ) insulin reduces the accumulation of liver cyclic AMP in the presence of glucagon. In this article we will present more recent observations on the interaction of glucagon, epinephrine, and insulin in the control of hepatic metabolism. The investigations have employed the isolated rat liver perfused by a modification of the technique of Mortimore.* The perfusion medium consisted of Krebs-Henseleit bicarbonate buffer containing 3 % bovine albumin and 20% bovine erythrocytes. Livers were from fed rats weighing 100-150 g and the perfusion flow rate was about 7 ml per minute. The perfusions were carried out in two ways. In most cases, livers were perfused for one hour with recirculating medium and hormones were infused into the portal vein at a constant rate. In these experiments, the hormone concentrations were calculated by dividing the quantity of hormone infused during the hour by the final volume of perfusate. Since this does not allow for degradation of hormone, the values are doubtlessly overestimated. In the second type of experiment livers were perfused initially for 20 minutes with recirculating media containing no additions in order to establish steady metabolic rates and levels of cyclic AMP. The perfusion system was then changed to one with nonrecirculating medium by diverting the perfusate leaving the liver into a beaker. After a six-minute control period, infusions of hormone were commenced and livers and effluent media were sampled at designated intervals. In these experi-